The j5R open reading frame (ORF) of the Malawi LIL 20/1 African swine fever virus (ASFV) isolate encodes a 111 amino acid protein with a putative transmembrane domain at the N terminus. Antisera raised against the predicted C-terminal peptide were used to identify the j5R protein by Western blotting in cells infected with ASFV or with recombinant vaccinia virus expressing the j5R ORF. This showed that the j5R protein migrates with an apparent molecular mass of 23-25 kDa, depending on the virus isolate, on SDS-PAGE and is expressed late during ASFV infection. The localization in infected cells and in virions of the j5R protein, and that of a previously characterized virion protein, j13L, which also contains a putative transmembrane domain, were studied by immunofluorescence and immuno-electron microscopy. Both proteins were expressed at 8-10 h post-infection (p.i.) as small multiple perinuclear foci which coalesced to a single area indicative of the virus factory at 18 h p.i. At the ultrastructural level j5R and j13L were detected mainly on membrane-like structures within the virus factory and on virus particles, suggesting that they may be involved in particle assembly. Negative contrast immuno-electron microscopy of mature extracellular virions confirmed that they are also integral structural proteins.